Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately following the Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art. The Sequence Listing is provided following the list of references and before the claims.
The present invention relates generally to synthetic, cyclic peptides for bacterial interference.
Staphylococcus aureus (S. aureus) is an important pathogen in humans which is now under increasing risk of developing antibiotic resistance to currently available therapeutics. Consequently, there is a pressing need to identify new types of antibiotic agents effective against these drug resistant bacterial strains. The phenomenon of xe2x80x98bacterial interferencexe2x80x99 may provide as yet unexplored avenues for the design of these new therapeutics. Bacterial interference refers to the ability of one organism to disrupt the biological functions of another. Until recently this survival process was thought to occur solely through a growth inhibition mechanism (Ji, G., et al., 1997), however a novel type of bacterial interference in S. aureus has been described which involves the inhibition of the so-called agr response (Novick, R. P., et al., 1993, Morfeldt, E., et al., 1995). This process is mediated by short secreted peptides containing a putative thiololactone ring structure. Chemical synthesis confirms that the native Agr peptides contain a thiololactone moiety, and that this structure is absolutely necessary for full biological activity. In addition, structure-activity studies are described by the present invention which offer insights into the nature of the agr activation and inhibition mechanisms.
Accessory genes allow bacteria to survive and multiply in plant or animal hosts. In S. aureus these virulence factors (cytotoxins and tissue-degrading enzymes) are under the control of the agr locus which contains two divergent promoters, P2 and P3. The RNA transcript from the P3 promoter is responsible for the upregulation of secreted virulence factors as well as the downregulation of surface proteins, the agr response (Novick, R. P., et al., 1993, Morfeldt, E., et al., 1995). There are four genes, agrA-D, in the P2 operon which code for the cytosolic, transmembrane and extracellular components of a density-sensing/autoinduction circuit (Novick, R. P., et al., 1995). The product of the agrD gene is a pro-peptide which is processed and secreted through AgrB, an integral membrane protein. The active AgrD peptide is then thought to bind to the transmembrane receptor coded by the agrC gene. Binding of the AgrD peptide triggers a standard two-component signal transduction pathway in which the AgrC receptor becomes autophosphorylated on a histidine residue leading to subsequent trans-phosphorylation of the AgrA gene product. Phosphorylated AgrA then activates the transcription from the P2 and P3 agr promoters (Novick, R. P., et al., 1995).
S. aureus strains can be divided into a least three groups (Ji, G., et al., 1997), each of whose secreted AgrD peptide can activate the agr response within the same group and inhibit the agr response in strains belonging to the other groups. It is the latter effect that constitutes a novel form of bacterial interference (Ji, G., et al., 1997). The AgrD autoinducing peptides, generated following processing and secretion through AgrB, consist of seven to nine residues. Interestingly, the sequences are highly variable among the groups, although all contain a conserved cysteine residue 5 amino acids from the C-terminus. Mass spectrometric analysis of AgrD peptides isolated from culture supernatants indicated a mass discrepancy of xe2x88x9218 Da compared to the predicted masses based on the peptide sequences (Ji, G., et al., 1995). This observation combined with the presence of the conserved cysteine residue in AgrD peptides, has led to the suggestion that these secreted peptides contain an intramolecular thiol ester linkage between the cysteine sulfhydryl group and the carboxy-terminus (Ji, G., et al., 1997). Consistent with this thiololactone structure, the addition of hydroxylamine to a purified AgrD peptide was observed to abolish its biological activity (Ji, G., et al., 1997).
The inability to isolate significant quantities of secreted AgrD peptides means that very little is known about the biochemistry of the AgrD/AgrC interaction. For example, the potency of the AgrD peptide in either activating (within S. aureus strains of the same group) or inhibiting (in S. aureus strains from other groups) the agr response is unknown. Equally, it is essential to determine whether the putative thiololactone structure within the AgrD peptides is required for activation of the agr response, inhibition of the agr response or both. The present disclosre provides such elucidation. This study detailed herein confirms the presence of the thiololactone moiety within the AgrD peptides through total chemical synthesis. Having demonstrated synthetic access to the system, more rigorous biochemical and structure-activity studies on the AgrD/AgrC interaction are addressed. The present disclosure further delineates that elimination of the thiol ester component of the cyclic ring structure can destroy activity activating the agr response while preserving (and enhancing) inhibitory activity.
The present invention provides a cyclic peptide comprising the structure: 
wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, R is selected from the group consisting of oxygen, nitrogen, sulfur and carbon, n is 0 to 10 and y is 1 to 10. The invention also contemplates a peptide composition comprising the provided cyclic peptide and a carrier.
The present invention also provides a cyclic peptide comprising the amino acid sequence of NH2xe2x80x94X(n)xe2x80x94Zxe2x80x94X(y)xe2x80x94COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10. The invention also contemplates a peptide composition comprising the provided cyclic peptide and a carrier, as well as therapeutic methods for treatment of infection that involve the administration of the pharmaceutical compositions that are and may be prepared in accordance with the teachings of the invention herein. Yet further, the invention extends to methods for the preparation of the cyclic peptide involving a cyclization protocol that is described in further detail herein and is illustrated in Example 1 and in FIG. 1A, and that itself is inventive.
Accordingly, it is a principal object of the present invention to provide a cyclic peptide comprising the amino acid sequence of NH2xe2x80x94X(n)xe2x80x94Zxe2x80x94X(y)xe2x80x94COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10.
It is a further object of the present invention to provide a cyclic peptide comprising the amino acid sequence of NH2xe2x80x94X(n)xe2x80x94Zxe2x80x94X(y)xe2x80x94COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10, that is devoid of activating activity and which retains inhibitory activity.
It is yet a further object of the present invention to provide a cyclic peptide comprising the amino acid sequence of NH2xe2x80x94X(n)xe2x80x94Zxe2x80x94X(y)xe2x80x94COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10, wherein the bond is a selected from the group consisting of a lactam ring and a lactone ring.
It is a still further object of the present invention to provide a pharmaceutical composition which comprises the provided peptides and a pharmaceutically acceptable carrier.
It is yet another object of the present invention to provide a method for the treatment of an S. aureus infection in a subject comprising administering to the subjects the provided pharmaceutical composition in an amount effective to treat the infection in the subject.
It is a still further object of the invention to provide methods for the preparation of the cyclic peptides of the invention that involve a solid phase cyclization protocol as illustrated and described herein.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.